The development of rhamnolipid production systems using non-pathogenic bacteria

Abstract

Rhamnolipids are amphiphilic surface active molecules that are currently receiving major interest from industry as a sustainable alternative to chemical surfactants produced using petrochemical feedstock’s. Whilst the majority of research has been carried out on P. aeruginosa in terms of industrial scale RL production the organisms’ pathogenic nature makes its use on an industrial scale uneconomic. The main focus of this thesis was therefore to explore whether B. thailandensis, a non-pathogenic alternative to P. aeruginosa, could be characterized and manipulated in order to produced RLs at a level equal to or greater than that of P. aeruginosa.

The first part of this thesis was used to characterize RL production in B. thailandensis and to gauge its potential as a possible industrial scale RL producer. As very little work has been carried out on B. thailandensis in terms of RL production this chapter was used to generate a detailed, quantitative outline of RL production in B. thailandensis under specific growth conditions as well as giving an insight into some of the phenotypic traits RLs are involved in. In addition, quantitative expression analysis of RL synthesis genes also provides a transcriptional profile of RL production providing a complete and in depth profile of RL biosynthesis in B. thailandensis E264.

The second part of this thesis was used to determine whether it was possible to increase the production of RL in B. thailandensis through genetic manipulation. This chapter identified a functional PHA synthesis system in B. thailandensis that competes for the same fatty acid precursors as RLs. A number of B. thailandensis mutants with transposon insertions in the identified PHA synthesis genes were screened for both increased RL production and subsequent decreased PHA production. The most significant increase in RL production was observed in the B. thailandensis phbB1 mutant which also showed a significant decrease in PHA production. Further analysis showed that knocking out PHA synthesis changed the ratio of mono-RL to di-RLs produced by B. thailandensis.

The final chapter in this thesis aimed to optimize the environmental and nutritional conditions for B. thailandensis fermentation to increase RL production during batch fermentation. A mineral salt medium (MSM) was used so that specific concentrations of nutrients could be controlled and results showed that RL production increased when B. thailandensis was grown under nitrogen and phosphate limitation. Sodium nitrate was also shown to be the best source of nitrogen for RL production compared to ammonium nitrate and other organic nitrogen sources. The source of carbon was also shown to be an important factor in RL production in B. thailandensis with RSO showing increased RL production when compared to glycerol. In addition this chapter showed that incubation temperature and seed culture volume can also affect overall RL production.

Overall this thesis outlines the biotechnological potential of B. thailandensis as an industrial scale RL producer and uses both genetic manipulation and process optimization methods to show how these systems can be used to increase RL production. In addition this thesis shows that B. thailandensis overcomes many of the problematic barriers identified in P. aeruginosa with regards to manipulating RL production for industrial applications. This work provides a significant contribution to the understanding of RL synthesis in B. thailandensis and presents a number of interesting findings that can be used as a base for further research.

Date of Award	Apr 2016
Original language	English
Supervisor	Ibrahim Banat (Supervisor) & Roger Marchant (Supervisor)